Abstract

Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure-induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans.

(A and B) Primary cardiomyocytes from neonatal rats were stimulated with saline or 30 μM PE in the presence of curcumin (10 μM in A and 5 or 10 μM in B) or a corresponding amount of its vehicle (DMSO) as a control for 48 hours. (A) These cells were subjected to immunocytochemistry using the primary antibody against cardiac MHC followed by staining with a secondary antibody conjugated with peroxidase (brown signals). Scale bar: 10 μm. (B) Myocardial cell-surface area was measured as described in Methods. Values in each group are mean ± SEM (μm) from 50 cells. (C and D) Cardiomyocytes were transfected with 0.5 μg of pANF-luc (C) or pβ-MHCluc (D) and 0.0025 μg of pRL-SV40. Then, these cells were stimulated with saline or 30 μM PE in the presence of curcumin or a corresponding amount of its vehicle for 48 hours. The relative promoter activities were calculated from the ratio of firefly Luc activity to sea pansy Luc activity. The data shown are mean ± SEM from 3 independent experiments, each carried out in duplicate.

(A and B) Cardiomyocytes were transfected with 0.7 μg of pCMVwtp300 (lanes 2 and 3) or pCMVβ-gal (lane 1) and incubated with curcumin (10 μM, lane 3) or a corresponding amount of its vehicle (lanes 1 and 2) for 48 hours. (A) Protein extracts from these cells were subjected to Western blotting using the anti-p300, anti-GATA4, and anti–β-actin antibodies. (B) Cell-surface area was measured as described in Methods. Values in each group are mean ± SEM (μm) from 50 cells. Cardiomyocytes were cotransfected with 0.5 μg of pANF-luc (C) or pβ-MHCluc (D), 0.0025 μg of pRL-SV40, and 0.25 μg of pCMVwtp300 (lanes 2 and 3) or pCMVβ-gal (lane 1). Then, these cells were incubated with curcumin (10 μM, lane 3) or its vehicle (DMSO, lanes 1 and 2) for 48 hours. The relative promoter activities were calculated from the ratio of firefly Luc to sea pansy Luc and expressed as the mean ± SEM from 3 independent experiments, each carried out in duplicate. (E) Cardiomyocytes were transfected with 0.7 μg of pCMVwtp300 (bars 2 and 3), pCMVHATmutp300 (bars 4 and 5), or pCMVβ-gal (bars 1, 6, and 7) and incubated with TSA (bars 6 and 7), curcumin (10 μM, bars 3, 5, and 7), or a corresponding amount of their vehicles for 48 hours. Cell-surface area was measured as described in Methods. Values in each group are mean ± SEM (μm) from 50 cells.

Primary cardiomyocytes from neonatal rats were stimulated with saline (lane 1) or 30 μM of PE (lanes 2 and 3) in the presence of curcumin (10 μM, lane 3) or a corresponding amount of its vehicle (DMSO, lanes 1 and 2) as a control for 48 hours.

(A) Proteins isolated by acid extraction from these cells were subjected to Western blotting for acetylated histone-3/4 or total histone-3/4 as indicated. (B) These cells were subjected to indirect immunofluorescence analysis with antibody against acetylated lysine. Original magnification, ×200. (C) Protein extracts from these cells were subjected to Western blotting with anti-GATA4 antibody, anti-p300 antibody, or anti–β-actin antibody. (D and E) The same extracts (100 μg of protein) were immunoprecipitated with goat anti-GATA4 polyclonal antibody (D) or rabbit anti-p300 polyclonal antibody (E) and subjected to sequential Western blotting with anti–acetylated lysine antibody, anti-p300 antibody, and anti-GATA4 antibody. (F–H) The amounts of GATA4-associated p300/total GATA4 binding (F), acetylated GATA4/total GATA4 (G), and p300-associated GATA4/total p300 binding (H) were quantified by densitometry with the use of Multi Gauge V3.0 (FUJIFILM). The data shown are mean ± SEM from 3 independent experiments.

(A and B) Primary cardiomyocytes from neonatal rats were stimulated with saline (lane 1) or 30 μM of PE (lanes 2 and 3) in the presence of curcumin (10 μM, lane 3) or a corresponding amount of its vehicle (DMSO, lanes 1 and 2) as a control for 48 hours. The nuclear extracts from these cells were probed with a radiolabeled double-stranded oligonucleotide containing the ET-1 GATA site (A) and with one containing the Sp-1 site (B). (C) The amount of GATA4/DNA binding (indicated by a long or short arrow) was quantified by densitometry with the use of NIH Image 1.61. The data shown are mean ± SEM from 3 independent experiments.

Curcumin inhibits hypertension-induced acetylation of GATA4 as well as p300/GATA4 complex in the LVs of Dahl rats.

(A) Protein extracts from the hearts of DR and DS rats at the age of 18 weeks were subjected to Western blotting with anti-p300 antibody, anti-GATA4 antibody, or anti-GAPDH antibody. (B) These extracts were then immunoprecipitated with goat anti-GATA4 polyclonal antibody and subjected to sequential Western blotting with anti–acetylated lysine antibody, anti-p300 antibody, and anti-GATA4 antibody. (C and D) Protein extracts from the hearts of DS rats treated with curcumin (50 mg/kg/d) or vehicle (1% gum arabic) for 7 weeks were subjected to Western blotting for p300, GATA4, and GAPDH (C) and then immunoprecipitated with anti-GATA4 antibody followed by sequential Western blotting for acetylated lysine, GATA4, and p300 (D). (E) The levels of signals for acetylated GATA4 and those for total GATA4 were quantified. Results are expressed as mean ± SEM from 8 vehicle-treated rats and 6 curcumin-treated rats.